G proteins 23 subunits play a central role in G-protein coupled receptor (GPCR)- mediated signal transduction. They act as cofactors in the receptor-mediated activation process as well as playing direct roles in signal transfer to downstream targets. Considerable data has accumulated in number of systems that excess 23 signaling has pathological consequences and that manipulation of 23 subunit signaling could be an effective therapeutic strategy in heart failure as well as other diseases. We have developed a novel targeting strategy for selective manipulation of G protein 23 subunit signaling pathways by selectively blocking 23-subunit binding interactions with functional protein partners using small molecules. In our preliminary data we demonstrate these compounds are efficacious in animal models of heart failure, inflammation and morphine-dependent analgesia. In the proposed experiments we will explore the fundamental mechanisms underlying binding and selectivity of these 23 binding compounds. In Specific aim 1 the experiments will characterize the selectivity of compounds for a broad range of G23 targets and develop new assays for predicting compound selectivity. Specific aim 2 will combine biophysical ligand interaction methods, mutagenesis and x-ray crystallography to develop a detailed understanding of the mechanisms for selectivity. Specific aim3 will exploit the information from specific aim 2 to refine our computational screening approach to allow for more efficient compound identification that may have unique mechanisms of action. Specific aim 4 will apply our knowledge of selectivity to GPCR signaling in neutrophil functions. Successful completion of the proposed experiments will lead to a thorough understanding of a the mechanism of action of a new family of molecules that target G23 signaling that have potential uses in dissecting the mechanisms of action of GPCR stimulated signaling and providing the basis for novel therapeutic approaches. G protein coupled receptors (GPCRs) are a major class of transmembrane receptors responsible for recognition of a large class of diverse ligands. Here we propose investigation of selective small molecule inhibitors of G protein 23 subunits identified in our laboratory which could be used to inhibit multiple GPCRs and modify actions of existing GPCR directed pharmaceuticals. Public Health Relevance: Results of these experiments will help to validate this alternate approach to modification of signaling pathways downstream of GPCRs that could ultimately lead to development of novel therapeutics.